Process for racemizing alpha-amino-epsilon-caprolactam

ABSTRACT

A-AMINO-E-CAPROLACTAM IS RACEMIZED BY A MICROBIOLOGICAL MEANS.

United States Patent 3,796,632 PROCESS FOR RACEMIZING a-AMINO- e-CAPROLACTAM Takashi Fukumura, Kamakura, Japan, assignor to Toray Industries, Inc., Tokyo, Japan No Drawing. Filed Dec. 10, 1971, Ser. No. 206,866

Int. Cl. C12d 13/06 US. Cl. 195-29 6 Claims ABSTRACT OF THE DISCLOSURE a-amino-e-caprolactam is racemized by a microbiological means.

DESCRIPTION OF THE PRIOR ART 'Racemization of a-amino-e-caprolactam by chemical or physicochemical methods is well known. However, no prior art is known in respect to microbiological racemization of a-amino-e-caprolactam.

SUMMARY OF THE INVENTION This invention relates to a new and useful method for racemizing optically active u-amino-e-caprolactam (hereinafter a-amino-e-caprolactam is referred to as aminolactam), by microbiological means, and to a method for preparing L-lysine quantitatively from D-L- or D-aminolactam by the racemization method of this invention in combination with a microbiological hydrolysis method.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 3,796,632 Patented Mar. 12, 1974 "ice activity of certain microorganisms. In accordance with this invention, the racemizing activity of the microorganisms belonging to the genera Achromobacter, Flavobacterium, and Alcaligenes, and the enzyme produced by said microorganisms will bring about the racemization of aminolactam, such as the conversion of D-aminolactam to DL-aminolactam.

The present inventor has isolated certain strains of bacteria from soil that have an aminolactam-racemizing activity, which had been neither known nor, to the best of the inventors knowledge, reported anywhere.

In detail, it has been found that D-aminolactam is converted into L-lysine when incubated in contact with a cell suspension of any one of several isolated bacterial strains and then, after heat treatment, incubated with a preparation having L-aminolactam-hydrolyzing activity, such as an acetone-dried cell preparation of Cryptococcus laurentii TORAY 2001, PERM-P 709.

Subsequently, Achrom'obacter cycloclastes IAM 1013 was found to have the same aminolactam-racemizing activity. The above described aminolactam-racemizing activity has also been found in a growing culture, a culture broth, an intact cell suspension, treated (or artificially modified) cells such as vacuum-dried cells and lyophilized cells, a cell extract, and an enzyme, namely aminolactam racemase.

The bacterial strain which is employed in the present invention can be chosen from strains of the genera to which the bacterial samples given the PERM-P 772, 776, 777, 778, 780 and IAM 1013 taxonomically belong.

(PERM-P is a Deposit Number in the Fermentation Research Institute in Japan.)

According to the present inventors classification, strains of PERM-P 772, 789 are Achromobacter cycloclastes, the strain of FERM-P 777 is F lavobacterium arborescens, the strain of FiE'RM-P 778 is Alcaligenes faecalz's, and the strain of FERM-P 776 is a new species, named by the inventor herein, Achromobacter obae sp. nov. Fukumura (197 1).

In Table 1, the characteristics of the PERM-P 772, 780, 776 and IAM 1013 are presented.

TABLE 1 FE RM-P 772 FE RM-P 780 FE RM-P 776 Achromobacter cucloclastea, Achromobacter cyclaclastes, Achromobacter cycloclastea, Achromobacter oboe sp. nov., Specific name IAM 1013 TORAY 1001 TORAY 1009 TORAY 1005 I. Microscopic observations:

(1) Shape of cells.... Rods, 1.0 x (1.8-2.0) n, Rods, 0.8 x (1.5-3.2) u, actively motile by means of perimotile by means of triehous flagella peritrichous flagella (2) Gram staining Negative II. Observations on cultures:

(1) Colonies on bouillonagar plate:

(1 Growth Moderate (2 Shape Circular or irregular, Circular, entire, convex, Circular, entire, umbonate,

undulate, umbonate smooth smoo (3) Qualities Glistening, butyrous Glistening Glistening, butyrous. (4) Color and trans- Non-pigment-torming whitish bufi, translucent with opaque center parency. (2) Streak culture on bouillon-slant:

(1) Growth Moderate, flliform Moderate, filiform, smooth Moderatg, filiform, slightly Moderatfi, filiiorm, slightly mucoi sprea mg. (2) Color Non-pigment-forming, whitish butt; translucent Non pigment-forming, Non-pigment-forming, light translucent, glistening greyish brown, medium darkened.

(3 Bouillon agar stab....- Surface growth, nail-head growth (4 Gelatin stab Surface growth, no liquefaction (5) Bouillon broth Slightly turbid, pellicle formation, sediment of pellicle (6) Potato Non-pigment-iorming, light greyish brown, raised growth, potato darkened Non-pigmentriorming, light grey-yellowish brown slightly spreading, potato darkened.

III. Growth conditions Good growth at 30 0. good growth at pH 7.0-7.5 aerobic, facilitative TABLE 1Continued FE RM-P 772 FE RM-P 780 FE RM-P 776 Achromobacter cycloclaates, Achro'mobacter cycloclastea, Achromobacter cycloclartes, Achromobacter obae sp. 1201)., Specific name 1AM 1013 TO RAY 1001 TORAY 1009 TORAY 1005 1X2} Physiological characteris- (1; Ammonia Produced (2 Indole. Not produced (3) Hydrogen Produced Remarkably produced.

(4) Acetylmethyl- Not produced carblnol. (5) Catalase Positive (6) Urease. Positive Negative.

(7) Nitrites Remarkably produced from nitrates Produced.

( itmus Alkaline after 2 or 3 days, no other detectable changes Shghtly alkaline after 8 days, no other detectable changes.

(9) Gelatin plate- No liquefaction (l0; Starch- Not hydrolyzed (11 Sugar fermentation- Neither acid nor gas produced from glucose, galactose, sucrose, maltose, and lactose (12) Litmus Not reduced in sugar fermentation media Slightly reduced n sugar fermentation media.

(13) L-Lysine utilizatlon.. (14) D and L-aminolactam utilization.

V. Source In Table 2, the characteristics of Ferm-P 777 are presented.

Utilized as carbon and nitrogen sources Utilized as carbon and nitrogen sources Isolated from soil TABLE 3-Contlnued FERMP 778, Alcaliaenes faecalis,

TABLE 2 TORAY 1007 FE RM P 777 II. Observations on cultures: Flavobacterium GTDOI'CJCMM, TO RAY g ggg g fii bgg gg 1006 (4) H No chromogenic, light yellowish brown, 1. Microscopic observations: 2 t k mt translucent with opaque center.

(1) Shape of cells Rods Q.7x1.7 ,noflagellum,non-motile l fig g ggg II. l se rv ai oil: %fitfires Negative. (1) Growth Moderate, filliorm, spreading, smooth. (1) colo ms on bouillon (2) Color Ll g hgt yellowish brown, opaque, gllstenarp a (1) Growth Moderate. gg%% f ?P-"-: i'fi g ilit g g (5) Bouillon broth. Turbid, sediment, no pellicle. g 3 n b in n (6) Potato Light brown, potatoes are darkened. 0 or 8Com g ye 111, Growth conditions Good growth at 30 0., good growth at 1 ghstemng' pH 7.0-7.5 aerobic. i gfif on boui IV Physiological properties (1) Growth Moderate, filii'orm. (2) fi s-$2 ga g ggfi Co o hlh y e llg lg becoming yellow, (3) Hydmh-g Produced opaque,g S g. (3) Boulllonagar stab Surface growth,nail-head growth. (4) l gfi Not produced (4) Gelatin stab Strat iiorln liquefaction, with yellow (5) gg Positive se lIIlQIl I 5 Bouillon broth Turbid, yellow sediment, n0 pellicle. fig gf ggfrom nitrates (6) Potato Abundant growth, wet and smooth surstrongly alkaline after (185m face, yellowish brownincolor, potatoes NO liquefaction m G th dm g i fi d th t (10) Starch Not hydrolyzedrow con OHS grow a H goo g a (11) Sugar iermentation-... Neither acid nor gas produced from PH gem faculta glucose, sucrose, maltose, lactose, 1V. Physiological and galactose.

charactenstlcs! P d d (12) Litmus Reduced in sugar fermentation media. 2 I d 3 d (13) L-Lysine utilization... Utilizedascarbon and nitrogen source. n 0 e 0 pro use (14) D and L-aminolactaln Do.

gydilogfig ifilfildeqfinin 11:10:21 proguced, later shghtly produced. utilization cey eycar norouce. (5) Catalase Positivm V. Source Isolated from soil.

Faintly positive. Not produced from nitrates. Slow peptopization, alkaline, latter beg ocld- These characteristics resemble that of Alcallgenes faengllfyirol zeo. calis 3160, and the descriptions in Bergeys Manual of Nelther acid nor g f g s Determlnatlve Bacteriology, 7th ed. $3521 sucrose ma ac ga As for the composition of the culture medium, either Litmus g' g in Sugar fermentation a synthetic ora natural culture medium is suitable so long 13 1.4 15) utilization mnized'ascarbon and nitrogen sources as it contamsthe essential nutrlents for the growth of the fz g f g bactcrlal straln employed and a small amount of D-, L-, v. Source lsolatedfromsoil, or DL-aminolactam as the inducer for the enzyme.

For instance, the medium employed in the examples of These characteristics resemble that Of Flavobactermm this invention consists of glucose pglypeparborescens IAM 1100 and the descrlptlons ln Bergeys tone, 05% Dbaminolactam hydrochloride, Manual of Determlnatlve Bacteriology, 7th ed. KzHPQb 33% KHzPOb 0 5% MgSO4.7H20, 02%

In Table 3, the characteristics of PERM-P 778 are 1 .5 and 5% yeast extract (PH 7.3) P Q Cultivation is carried out aerobically at 20-40 C., pref- TABLE 3 erably 32 C.

FERM-P 773, Alcaligme faecalig, As for the state of the aminolactanr-racemizing agent TORAY 1007 when used in the process of this inventlon, growing cells, I. Microscopic observations: a culture broth, an intact cell suspension, treated cells (1) Shape cells ggg gg gg -gg gg by means such as lyophilized cells and vacuum-dried cells, a cell 2) Gram staining Negative, extract, and an enzyme, namely aminolactam racemase, II. Observations on cultures: are n effective (1) Colonies on bouillon agaspgtevth M d t Moreover, the enzyme can be also used in the state of 1'0 0 era 0, P 1! (2) Shape Circular pulvjnate spreading un. lnsolublllzed enzyme such as DEAE-s TEAE': l'

dulate: lulose, DEAE-sephadex-adsorbed enzyme. (3) Quality Butymus' The aminolactam racemase, which is the s b tan f the aminolactam-racemizing agents, is characterized as follows:

(1) Catalytic action: racemize D- or L-aminolactam to optically inactive DL-aminolactam.

(2) Substrate specificity: does not act on D- or L-lysine.

(3) Active range of pH: active at pH 6-10, optimum at (4) Assay method: incubated with D-aminolactam aqueous solution of pH 8.5 and, after heat treatment, L- aminolactam amidohydrolase (such as acetone-dried cells of Cryptococcus laurentii TORAY 2001) is added and incubated. The amount of L-lysine produced in the mixture is analyzed.

(5) Optimum temperature: 50 C. for the incubation of about 30 minutes; 38-43 C. for the incubation of about 4 hours 33 C. for the incubation of about hours.

(6) Inhibitor: Hydroxylamine.

(7) Activator: Pyridoxal-5-pho,sphate.

(8) Purification: Fractionation with ammonium sulfate.

Fraction of about 35-40% saturation.

Microorganisms other than Achromobacter, Flavobacterium, and Alcaligenes may also be employed as producers of the aminolactam-racemizing agent so long as they produce the same aminolactam racemase as specified above.

In connection with the reaction step using the aminolactam racemizing agent, the preferred conditions are as follows.

The reaction mixture is composed of a proper amount of an aminolactam-racemizing agent and an aqueous so- L-lysine in substantially pure form, i.e., free of contamination with the D-lysine, independently of the optical property of the aminolactam used as the starting material. Thus, L-lysine is produced directly from DL-aminolactam. The L-aminolactam-hydrolyzing agent to be used in conjunction with the aminolactam-racemizing agent is preferably produced by the microorganisms belonging to the general Cryptococcus, Candida, and Trichosporon.

As for the state of the L-aminolactam-hydrolyzing agent when applied in the above case, growing cells, a culture broth, an intact cell suspension, treated cells such as lyophilized cells and acetone-dried cells, a cell extract, and an enzyme, L-aminolactam amidohydrolase, are effective.

L-aminolactam-hydrolyzing agent is used in an amount preferably of 0.1-10 weight percent of the aminolactam in the dry state.

The typical strains of the microorganisms belonging to Cryptococcus are strains of FERM-P 709, 710, Cryptococcus laurentii IFO 0609, IFC 0906, those belonging to Candida are strains of 'FERM-P 715, 717, 719, Candida humicola IFO 0753 (=ATCC 9949) and those belonging to Trichosporon are strains of FERM-P 712, 714, 725, Trichosporon cutaneum IFO 0173.

According to our classification, strains of PERM-P 709 and 710 were found to be Cryptococcus laurentii, strains of FERMP 715, 717 and 719 were Candida' humicola, and strains of FERM-P 712, 714 and 725 were Trichosporon cutaneum.

In Table 1a characteristics of the FERM-P 709, 710 and Cryptococcus laurentii IFO 0609, 0906 are presented.

TABLE 12.

FE RM-P 709 FE RM-P 710 Specific name C'rz/ptococcua laurentii, IFO Cryptococcus laurentii, IFO C'ryptococcus laurentii, TO- Cryptococcue laurentii, TO-

0609 0900 RAY 2001 RAY 2002 (3) Ascospore formation... II. Cultural characteristics:

Oval, (as) x (ta-9),.

Creeping 4. Ring Absent YM-egar colonies: 1. S

Smooth, dull Butyrous Light reddish yellow 6. C or-.- III. Physiological characteristics:

Round, oval long oval, elongate Round, oval, long oval Bound to oval, (2. x (ii-5); Multilateral budding Cells present, true myeelium, psuedomycelium, blastospores, ehlamydospores, arthrospores are not observed Not observed on a gypsum block or a modified Gorodkowa-egar in 30 days incubation N ct turbid Compact Occasionally creeping Occasionally very weak ring Circular Entire Convex Smooth, glistening ucous Light cream-colored (1) Sugar fermention N 0 gas produced irom lucose, galactose, sucrose, maltose, lactose, and raifinose in 30 days incubation (2) Sugar assimilation (auxanographic method).

(3 KNO3 assimilation...-

(4 Litmusmilk:

1. Color 2. Coagulation 3. Peptonization (5) Splitting oi arbutin--- (6; Starch-like compound (7 L-Lysineutilization..-

(8) L-Aininolactam utilization.

(9) Growth conditions..-.-

IV. Source Positive (violent reaction by lugol solution) Utilized as carbon and nitrogen sources Utilized as carbon and nitrogen sources Good growth at 30 0., good growth at pH 6.0

lucose, galactose, sucrose, maltose, lactose and raifinose are assimilated Absent (both on liquid culture and auxanographic method) Slightly aid, after 29 days reduced oaguleted after 26 days N 0 Positive (strong) Positive (blue reaction by lugol solution) Isolated from soil.

lution of 5-10 weight percent D- or L-aminolactam, pH 640, preferably pH 7.5-8.5.

The aminolactam racemizing agent is used preferably in an amount of 0.1-10 weight percent of the aminolactam in the dry state.

The reaction mixture is incubated at from room temperature to C., preferably with gentle stirring.

When the insolubilized enzyme is employed, it is preferably used in column form.

Moreover, the aminolactam racemization of this invention may be carried in the presence of an L-aminolactam-hydrolyzing agent, whereby the product is always 75 are TABLE 2a ATCC 9949 FERM-P 717 FERM-P 715 FERM-P 719 Candida humicola, TORAY Candida humicola, TORAY Candida humicola, TORAY Specific nam C L IFO 0753 2010 2008 2020 1. Microscopic observations:

(1) Cells in YM broth:

1. Shape Variable shape:round, lemon-shaped, oval, elliptical, elongate,cylindrical 2. Dominant cells Bound to oval, (3-4) x 7, Bound to oval, (5-6) x (5-9)p. Elliptical, (4-5) 1: (710) 3. Vegetative repro- Multilateral budding duction. (2) Slide culture on potato toagar:

1. Cells Present 2. True mycelium- Present Development. 3. Pseudomycelium..- Rich development 4. Blastospores Development 5. Chlamydospores- Not observed Present. 6. Arthspores Absent (3) Ascospore formation. Not observed on a gypsum block or a modified Gorodkowa-agar in 30 days incubation 11. Cultural characteristics:

. Not turbid Compact Flocculent. Islets to thin pellicle No formation (2) Clear ring Obscure ring Circular, becoming filamentous Entire, becoming filamentous Convex, at times umbonate- Convex Slightly umbonate Convex, later umbonete.

Butyrous, letter mucoid Butyrous (3) Yellowish Light cream-colored Abundant Moderate Abundant Filamentous Entire Undulate Filamentous. wrinkled, lusterless Smooth, dull Rough, dull Rough and wrinkled, dull. IIl'flPgysiological characs cs: (1) Sugar iermentation- No gas produced from glucose, galactose, sucrose, maltose, lactose and rafiinose in 30 days incubation (2) Sugar assimilation Glucose, galactose, sucrose, maltose, lactose are assimilated (auxanographic meth- Absent (both on liquid culture and auxanographic methods) Slightly red, later reduced Unchanged, after 22 days reduced Co ted Coagulated after 25 days 3. Peptonization Slow peptonization oi the coagulation After 30 days slow peptonization oi the coagulation (5) Splitting of arbutin. Positive (6; Starch like compound. Slightly produced or faintly produced Not produced. (7 L-Lysine utilization-.- Utilized as carbon and nitrogen sources lzi-Aminolactam utili- Utilized as carbon and nitrogen sources za on. (9) Growth conditions.-.- Good growth at 30 C. Good growth at pH 6.0

IV. Source Isolated from soil It is satisfactory to consider the three strains of Taxonomic Study (1967) and the close resemblance to FERM-P 715, 717, 719 as Candida and more particularly Candida humicola IFO 0753. Candida humicola on the basis of the description in J. 45 In Table 3a, characteristics of the FER'M-P 712, 714, Lodder and N. J. W. Kroger-Van Rijs The Yeasts, A 725 and T richosporon cutaneum I FO 0173 are presented.

TABLE 3a FE RM-P 714 FE RM-P 725 FE RM]? 712 Trlchosponm cutaneum, Trichosporon cutaneum, Trichosporon cutaneum, Trichasporon cutaneum, Specific name IFO 0173 TORAY 2007 TO RAY 2035 'IORAY 2004 I. Microscopic observations:

(1) Cells in YM broth:

1. Shape Variable shape; round, oval, long oval cyhndrical occasionally giant cells 2. Dominand cells-... Bound to oval, (e-a 840).. Lon oval, (4-5) x (740 Cylindrical. (7-s) M (20-30 3. Vegegative Maltilateral budding and fission Mainly fission.

repro uction. (2) Slide culture and Cells present, rich development of true mycelium, no pseudomycelium, blastospores round to oval forming clusters,

otato-agar. arthrospores in typical zigzag form (3 Ascospore formation. Not observed on gypsum block or a modified Gorodkowa-agar in 30 days incubation II. Cultural characteristics:

) YM b t Not turbid Flocculent Compact.

Pellicle formed, later going down as sediment Circular becoming filamentous Entire, becoming filamentous Filamentous. 3. Elevation Convex Convex, later umbilicate Raised. 4. Surface Rough, dull Smooth becoming rough, Smooth, becoming velvet- Wrinkled, lusterless.

dull like folded, lusterless 5. Quality Butyrous to dry Butyrous ry 6. Color Cream-colored Cream to whitish creamcolored. (3) YM slant culture:

1. Growth Abundant Moderate Abundant 2. Margin Filamentous Undulate, becoming fila- Undulate to filamentous mentous on the upper part 3. Surface (30 days ,Velvet-like, folded Rough Velvet-like, folded Wrinkled.

culture) TABLE 3aContinued FE RM-P 714 FE RM-P 725 FE RM-P 712 Trichosporon cutaneum, Trichosporon cutanemn, Trichosporon cutaneum, Trz'chosporon cutaneum, Specific name IFO 0173 TORAY 2007 TO RAY 2035 TORAY 2004 III. Physiological characteristics:

(1) Sugar fermentation..." (2) Sugar assimilation (auxanographic method) (3) KNOa assimilation- (4) Litmus milk:

1. Color No gas produced from glucose, galactose, sucrose, maltose, lactose, rafiinosc in 30 days incubation Glucose, galactose, sucrose, maltose, lactose are assimilated Absent (both on liquid culture and auxanographic methods) Unchanged, after 25 days reduced 2. Coagulation Coagulated after 25 days I 3. Peptonization No Slow peptonization of the No.

coagulation (5) Splitting of arbutin- Negative Positive Negative.

Not produced (6 Starch-like compound. 27 L-lysine utilization- 8 L-aminolactam utilization. (9) Growth conditions IV. Source Utilized as carbon and nitrogen sources Utilized as carbon and nitrogen sources Isolated from soil It is satisfactory to consider the three strains of FERM-P 712, 714, 725 as Trichosporon and more particularly Trichosporon cutaneum on the basis of the description in J. Lodder and N. J. W. Kroger-Van Rijs The Yeasts, A Toxonomic Study (1967) and the close resemblance to Trichosporon cutaneum IFO 0173.

As for the composition of the culture medium of the yeasts, either a synthetic or a natural culture medium is suitable so long as it contains the essential nutrients for the growth of the yeast strain employed, and a small amount of L- or DL-amino-lactam as the inducer for the enzyme. For instance, the medium employed in the examples of this invention consist of 1% glucose, 0.3% NH N'O 5% L- aminolactam hydrochloride, 0.1% KH PO 0.05% MgSO -7H O, 0.02% MnCl -5H O and 0.05 yeast extract (pH 6).

Cultivations of the yeasts are carried out aerobically at -40" C., preferably 30 C.

As for the state of the L-aminolactam-hydrolyzing agent when applied in the process of this invention, growing cells, a culture broth, living cells, modified cells such as lyophilized cells and acetone-dried cells, a cell extract, and a purified protein derived from cells, namely L-amino-lactam amidohydrolase are efiectively used. Moreover, the enzyme can be also used in the state of an insolublized enzyme such as DEAE-, TEAE-, GE-cellulose-adsorbed enzyme and DEAE-sephadex-adsorbed enzyme. Furthermore, as for the L-aminolactamhydrolyzing agent, the L-aminolactam amidohydrolase the characteristics of which are described in the next paragraph is specified. Therefore, microorganisms other than Cryptococcus, Candida and Trichosporon are also employed as producers of the L-aminolactam-hydrolyzing agent so far as they produce the same L-aminolactam amidohydrolase as specified in the present invention.

The characteristics of the L-aminolactam amidohydrolase are as follows:

(1) Catalytic action: hydrolyze L-aminolactam, producing L-lysine.

(2) Substrate specificity: (1) may hydrolyze D-aminolactam at a rate of 0.l-0.5% or so of the activity on L-amino-lactam; (2) not hydrolyze u-butyrolactam, fi-valerolactam, e-caprolactam, cyclic dimer, trimer and pentamer of e-aminocaproic acid, D and L-pyr- (4) Assay method: incubated with L-amiuolactam solution of a pH 9.0. The amount of L-lysine produced in the incubated mixture is analyzed.

(5) Optimum temperature: C. for an incubation of about 20 minutes. 40-50 C. for an incubation of several hours.

(6) Inhibitor: Ethylenediaminetetraacetic acid ('EDTA).

(7) Activator: Mn++, Mg++, Zn++, Co:

(8) Purification: Fractionation with ammonium sulfate. Fraction of about 30-40% saturation. Then, liquid chromatography using DEAE-cellulose.

DL-aminolactam, produced in the reaction mixture of the present invention, can be separated in the following way. The reaction mixture is centrifuged and a supernatant liquid is obtained. A small amount of activated charcoal is added to this supernatant liquid. The mixture is boiled for a few minutes, cooled to room temperature and filtered. The pH of the filtrate is adjusted with aqueous solution of HCl to pH 1. The solution is dried and a residue is obtained.

The residue is composed principally of aminolactam hydrochloride. The residue is suspended in a small amount of ethyl alcohol. The suspension is stirred for several hours at room temperature, and filtered. Crystals of aminolactam hydrochloride are obtained.

The following are several examples of the present invention.

Example 1 obtained from one hour and 20 hours reactions respec-- tively. The specific activity is expressed in ,umoles of L- amino-lactam produced/hour/mg. of dried cells. The conversion rate is expressed in rolidon oarboxylic acid. 65 X percent (3) Active range of pH: active at pH 6-12, being optimum at about pH 9.0.

TABLE 4 Conversion Specific r e Strain activity (percent) Achr IAM 1013 5.0 50 Achromobacter cycloclastes 5. 2 60 Achromobacter cycloclastes 2. 7 48 Flavobacterium arboresce'n 0. 8 25 Alcaligenes faecalis TORAY 1007, FE RM-P 778.- 2. B 47 Achromobacter obae sp. nov TORAY 1005, FE RM-P 776 3. 1 49 1 1 Example 2 Achramobacter cycloclastes TORAY 1001, FERM- P 772 was cultured aerobically in SL of the above described medium. Sixty grams of living cells were obtained from the culture.

The reaction mixture was composed of grams of the living cells, 10 grams of D-aminolactam and water (up to 100 ml.); pH was adjusted to 8.5 with HCI. Incubation was carried out at 40 C. for 10 hours.

From the above reaction mixture, 12.2 g. of DL-aminolactam hydrochloride was obtained.

Example 3 A grams aliquot of the living cells obtained in Example 2 was lyophilized, and 3.5 grams of dried cells were obtained.

The reaction mixture was composed of 3.5 grams of the dried cells and 100 m1. of 10% D-aminolactam aqueous solution of pH 8.5.

Incubation was carried out at 40 C. for 10 hours.

From the reaction mixture, 12.4 g. of DL-aminolactam hydrochloride was obtained.

Example 4 A 15 gram aliquot of the living cells obtained in Example 2 was suspended in the same volume of water. The suspension was subjected to a French-press, and was centrifuged.

Sixty ml. of a supernatant obtained was dialyzed to 10- M phosphate butfer at pH 8.5.

The reaction mixture was composed of the dialyzed supernatant liquid, 3 grams of L-aminolactam and 40 ml. of water, pH 8.5.

Incubation was carried out at 40 C. for 10 hours. From the reaction mixture, 3.7 g. of DL-aminolactam hydrochloride was obtained.

Example 5 Fifty-five grams of living cells of Achromobacter cycloclastes TORAY 1001, FERM-P 772 was obtained in the same way as in Example 2. The living cells were suspended in 50 ml. of l0- M phosphate buffer of pH 8.5. The suspension was subjected to a French-press and centrifuged. One hundred ml. of a supernatant liquid thus obtained was fractionated with ammonium sulfate. Each fraction was dialyzed against the phosphate buffer.

A fraction of 35-40% saturation comprised 48 ml. in volume and showed a specific activity of 54.6 ,umoles/ hr./ml. Into the enzyme solution (48 ml.), DEAE-cellulose (1 gram in dry weight) bufferized with 10- M phosphate buffer at pH 7.6, was soaked and filtered. The enzyme activity in the solution was completely adsorbed onto the DEAE-cellulose.

A column, 1 cm. 4; x cm., was made of the enzymeadsorbing DEAE-cellulose. Six hundred ml. of 0.5% D-aminolactam aqueous solution, pH 8.5, was allowed to flow through the column at 42 C. for 15 hours.

From the eflluent 3.5 g. of DL-aminolactam hydrochloride was obtained.

Example 6 Lyophilized cells of Achrom-obacter cycloclastes TORAY 1001, FERM-P 772 were prepared in the usual manner. The specific activity of the aminolactam racemization of this preparation was 4 imoles/hn/mg.

Acetone-dried cells of Cryptococcus laurentii TORAY 2001, FERM-P 709 were prepared in the usual manner. This preparation had L-aminolactam-hydrolyzing activity; and the specific activity of this preparation was 10 moles/hL/mg.

The reaction mixture was composed of 600 mg. of the lyophilized cells of Achromobacter cycloclastes TORAY 1001, 200 mg. of the acetone-dried cells of Cryptococcus laurentii TORAY 2001, 3 grams of D- aminolactam and ml. of water (pH 8.5).

Incubation was carried out at 40 C. for 24 hours.

'From the reaction mixture, 4.15 grams of L-lysine monochydrochloride was obtained. The yield from D- aminolactam was 97%. The optical purity was 99%.

Example 7 Lyophilized cells of Alcaligenes faecalis TORAY 1007, PERM-P 778 were prepared in the usual manner. The specific activity of aminolactam racemization of this preparation was 2 ,umoles/hn/mg.

Acetone-dried cells of Candida humicola TORAY 2010, PERM-P 717 were prepared in the usual manner. This preparation had a L-aminolactam-hydrolyzing activity, and the specific activity was 6 nmoles/hn/mg.

A reaction mixture was composed of 600 mg. of the lyophilized cells of Alcaligenes faecalis TORAY 1007, 300 mg. of the acetone-dried cells of Candida humicola TORAY 2010, 3 g. of DL-aminolactam and 30 ml. of Water (pH 8.5).

Incubation was carried out at 40 C. for 24 hours.

From the reaction mixture, 4.07 g. of L-lysine monohydrochloride was obtained. The yield from DL-amino lactam was The optical purity was 98%.

I claim:

1. A process for racemizing a-amino-e-caprolactam which comprises subjecting optically active u-amino-ecaprolactam to the action of a microorganism having aminolactam-racemizing activity selected from the genera Achromobacter, Flavobacterium, and Alcaligenes.

2. A process as recited in claim 1 wherein DL-a-aminoe-caprolactam so produced in the reaction mixture is then isolated in a conventional manner.

3. A process as recited in claim 1 wherein DL-a-aminoe-caprolactam so produced in the reaction mixture is subjected to a subsequent reaction consisting of the asymmetrical hydrolysis of a-amino-s-caprolactam.

4. A process for racemizing a-amino-e-caprolactam which comprises contacting optically active at-BIIliIlO-e caprolactam in an aqueous solution with a racemization agent consisting of the enzyme, aminolactam racemase, which is isolated from cells of microorganisms having aminolactam-racemizing activity selected from the genera Achromobacter, Flavobacterium and Alcaligenes, and subjecting said caprolactam to the racemizing action of said racemization agent.

5. A process for racemizing a-amino-e-caprolactam in an aqueous solution which comprises subjecting optically active a-amino-e-caprolactam to racemization by the catalytic action of a member of the group consisting of growing culture, a culture broth, living cells, dried cells or a cell extract of a microorganism, selected from the genera Achromobacter, Flavobacterium, and Alcaligenes, by contact therewith, said member having aminolactam racemizing activity.

6. A process for preparing L-lysine which comprises subjecting a-amino-e-caprolactam in an aqueous solution to the action of a microorganism having aminolactamracemizing activity selected from the genera Achromobacter, Flavobacterium, and Alcaligenes or the enzyme, aminolactam racemase, which is isolated from cells of said microorganism, in the presence of a microorganism having L-aminolactam hydrolyzing activity selected from the genera Cryptococcus, Candida, and Trichosporon or L-aminolactam amidohydrolase which is isolated from cells of said L-aminolactam hydrolyzing microorganism and recovering L-lysine so produced from the reaction mixture.

References Cited UNITED STATES PATENTS 3,056,729 10/196 2 Seto -29 ALVIN E. TANENHOLTZ, Primary Examiner 

